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(11) | EP 3 246 339 B1 |
(12) | EUROPEAN PATENT SPECIFICATION |
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(54) |
ANTIBODIES TO BRADYKININ B1 RECEPTOR LIGANDS ANTIKÖRPER GEGEN BRADYKININ-B1-REZEPTOR-LIGANDEN ANTICORPS AUX LIGANDS DU RÉCEPTEUR DE LA BRADYKININE B1 |
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Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). |
SEQUENCE LISTING
BACKGROUND
SUMMARY
X1 is Y, F or H,
X2 is R, D, A, V, L, I, M, F, Y or W,
X3 is Y, F, W or H,
X4 is D, E or Y, and
X5 is D or E;
X1 is W or F,
X2 is N or no amino acid,
X3 is Y or S,
X4 is D or P, and
X5 is F or Y;
h) SEQ ID NO: 8 [YFX1PX2NGNTGYNQKFRG], wherein
X1 is D, R, A, V, L, I, M, F, Y or W, and
X2 is Y, D, E, N, or Q;
i) SEQ ID NO: 64 [WX1DPENGDX2X3YAPKFQG], wherein
X1 is I, or V,
X2 is T, or S, and
X3 is G, or D;
j) SEQ ID NO: 14;
k) SEQ ID NO: 33;
l) SEQ ID NO:41;
m) SEQ ID NO: 48; and
n) SEQ ID NO: 56;
o) SEQ ID NO: 9 [GYSFTDYX1IY], wherein X1 is N, W or Y;
p) SEQ ID NO: 65 [GFNIKDYYX1H], wherein X1 is L, or M;
q) SEQ ID NO: 15;
r) SEQ ID NO: 34;
s) SEQ ID NO: 42;
t) SEQ ID NO: 49; and
u) SEQ ID NO: 57;
v) SEQ ID NO: 10 [QQ X1 X2S X3P X4T], wherein
X1 is Y, F or H,
X2 is Y, F, H or W,
X3 is Y, F, T or H, and
X4 is W, Y, F, H or L;
w) SEQ ID NO: 66 [QX1X2X3SX4PX5T], wherein
X1 is Q or N,
X2 is Y, F, D or H,
X3 is Y, F, H or W,
X4 is Y, F, T or H, and
X5 is W, Y, F, H or L;
x) SEQ ID NO: 69 [X1QGTHFPYT], wherein X1 is L or M;
y) SEQ ID NO: 16;
z) SEQ ID NO: 35;
aa) SEQ ID NO: 43;
bb) SEQ ID NO: 50; and
cc) SEQ ID NO: 58, or
dd) SEQ ID NO: 11 [WASTRX1], wherein X1 is E, D, Q or N;
ee) SEQ ID NO: 67 [X1ASTRX2], wherein
X1 is W or G, and
X2 is E, D, Q or N;
ff) SEQ ID NO: 17;
gg) SEQ ID NO: 36;
hh) SEQ ID NO: 51; and
ii) SEQ ID NO: 59;
andjj) SEQ ID NO: 12 [KSSQSLL X1SSNQKN X2LA], wherein
X1 is W, H, Y or F, and
X2 is H or Y;
kk) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
X1 is W, H, Y or F,
X2 is S or G,
X3 is N or D,
X4 is K or R,
X5 is H or Y;
ll) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN],wherein X1 is K or E;
mm) SEQ ID NO: 18;
nn) SEQ ID NO: 37;
oo) SEQ ID NO: 44;
pp) SEQ ID NO: 52; and
qq) SEQ ID NO: 60.
X1 is Y, F or H,
X2 is R, D, A, V, L, I, M, F, Y or W,
X3 is Y, F, W or H,
X4 is D, E or Y, and,
X5 is D or E;
X1 is W or F,
X2 is N or no amino acid;
X3 is Y or S,
X4 is D or P, and
X5 is F or Y;
X1 is D, R, A, V, L, I, M, F, Y or W, and
X2 is Y, D, E, N, or Q;
X1 is I, or V,
X2 is T, or S, and
X3 is G, or D;
X1 is Y, F or H,
X2 is Y, F, H or W,
X3 is Y, F, T or H, and,
X4 is W, Y, F, H or L:
X1 is Q or N,
X2 is Y, F, D or H,
X3 is Y, F, H or W,
X4 is Y, F, T or H, and
X5 is W, Y, F, H or L;
X1 is W or G, and
X2 is E, D, Q or N;
a) SEQ ID NO: 12 [KSSQSLL X1SSNQKN X2LA], wherein
X1 is W, H, Y or F, and
X2 is H or Y;
b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
X1 is W, H, Y or F,
X2 is S or G,
X3 is N or D,
X4 is K or R,
X5 is H or Y.
c) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN],wherein X1 is K or E;
b) SEQ ID NO: 18;
c) SEQ ID NO: 37;
d) SEQ ID NO: 44;
e) SEQ ID NO: 52; and
f) SEQ ID NO: 60.
X1 is Y, F or H,
X2 is Y, F, H or W,
X3 is Y, F, T or H, and,
X4 is W, Y, F, H or L:
X1 is Q or N,
X2 is Y, F, D or H,
X3 is Y, F, H or W,
X4 is Y, F, T or H, and
X5 is W, Y, F, H or L;
X1 is W or G, and
X2 is E, D, Q or N;
a) SEQ ID NO: 12 [KSSQSLL X1SSNQKN X2LA], wherein
X1 is W, H, Y or F, and
X2 is H or Y;
b) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
X1 is W, H, Y or F,
X2 is S or G,
X3 is N or D,
X4 is K or R,
X5 is H or Y.
c) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN],wherein X1 is K or E;
b) SEQ ID NO: 18;
c) SEQ ID NO: 37;
d) SEQ ID NO: 44;
e) SEQ ID NO: 52; and
f) SEQ ID NO: 60.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the results of ELISA assays demonstrating binding of EE1 antibody to kinin peptides.
Figure 2 depicts the results of differential scanning calorimetry measurements of antibody F151.
Figure 3 depicts amino acid sequence alignments of the variable regions of the murine and humanized F151 antibody. All identical residues are listed in the alignment, while homologous residues are identified by "+" sign and non-homologous residues are left blank. Figure discloses SEQ ID NOS 19, 24, 26, and 30, respectively, in order of appearance.
Figure 4 depicts an electron density map of the antigen binding site of the F151 antibody/kallidin complex.
Figure 5 depicts an electron density map of the antigen binding site of the F151 antibody/des-Arg10-kallidin complex.
Figure 6 depicts a ribbon and stick representation of the Fv subunit of F151 bound to kallidin.
Figure 7 depicts an amino acid sequence alignment of the light chain variable regions of exemplary murine anti-kallidin antibodies of the disclosure. Amino acid residues that interact with kallidin are marked with asterisks. Figure discloses SEQ ID NOS 134, 125, 124, 123, 126, and 131, respectively, in order of appearance.
Figure 8 depicts an amino acid sequence alignment of the heavy chain variable regions of exemplary murine anti-kallidin antibodies of the disclosure. Amino acid residues that interact with kallidin are marked with asterisks. Figure discloses SEQ ID NOS 135, 120, 119, 118, 121, and 122, respectively, in order of appearance.
Figure 9 depicts the results of in vivo experiments determining the effect of EE1 antibody on formalin-induced acute inflammatory pain.
Figure 10 depicts the results of in vivo experiments determining the effect of EE1 antibody on CFA-induced mechanical hypersensitivity.
Figure 11 depicts the results of in vivo experiments determining the effect of EE1 antibody on CFA-induced thermal hypersensitivity.
Figure 12 depicts the results of in vivo experiments determining the effect of EE1 antibody on CCI-induced mechanical hypersensitivity.
Figure 13 depicts the results of in vivo experiments determining the effect of EE1 antibody on CCI-induced thermal hypersensitivity.
Figure 14 depicts schematic maps of VL and VH expression constructs for generating humanized F151 variant HC3a/LC3a with the restriction DNA endonuclease sites presented as deduced sequences in bold and underlined. Panel A depicts the light chain and Panel B depicts the heavy chain. Figure discloses SEQ ID NOS 30, 24, and 136, respectively, in order of appearance.
Figure 15 depicts an alignment of the F151 heavy chain (A) and light chain (B) amino acid sequences with the closest human germline amino acid sequences.
Figure 16 depicts an alignment of the F151 heavy chain (A) and light chain (B) with a heavy chain locus (1-08 & 1-18) and light chain (V IV-B3) locus of the VH1 sub-family. CDR regions and Vernier regions are indicated in boldface and humanizing mutations are underlined.
Figure 17 depicts (A) the secondary and (B) tertiary structure of the main chain polypeptide backbone conformation of kallidin (KD) as bound to F151 antibody which comprises a type II tight turn at Proline 4 (C). Figure discloses SEQ ID NOS 2, 2, and 133, respectively, in order of appearance.
DETAILED DESCRIPTION
I. Definitions
II. Anti-Kallidin or des-Arg10-Kallidin Antibodies
Antibody Clone | Sequence | SEQ ID NO. |
F151 HCDR3 consensus | X1YX2X3DX4HAMX5Y | 7 |
where: | ||
X1 is Y, F or H; | ||
X2 is R, D, A, V, L, I, M, F, Y or W; | ||
X3 is Y, F, W or H; | ||
X4 is D, E or Y; and | ||
X5 is D or E. | ||
F151 HCDR2 consensus | YFX1PX2NGNTGYNQKFRG | 8 |
where: | ||
X1 is D, R, A, V, L, I, M, F, Y or W; | ||
and | ||
X2 is Y, D, E, N, or Q. | ||
F151 HCDR1 consensus | GYSFTDYX1 IY | 9 |
Where X1 is N, W or Y. | ||
F151 LCDR3 consensus | QQX1X2SX3PX4T | 10 |
where: | ||
X1 is Y, F or H; | ||
X2 is Y, F, H or W; | ||
X3 is Y, F, T or H; and | ||
X4 is W, Y, F, H or L. | ||
F151 LCDR2 consensus | WASTRX1 | 11 |
where X1 is E, D, Q or N. | ||
F151 LCDR1 consensus | KSSQSLLX1SSNQKNX2LA | 12 |
where: | ||
X1 is W, H, Y or F; and | ||
X2 is H or Y. | ||
F151 HCDR3 | YYRYDDHAMDY | 13 |
F151 HCDR2 | YFDPYNGNTGYNQKFRG | 14 |
F151 HCDR1 | GYSFTDYNIY | 15 |
F151 LCDR3 | QQYYSYPWT | 16 |
F151 LCDR2 | WASTRES | 17 |
F151 LCDR1 | KSSQSLLYSSNQKNYLA | 18 |
F151 VH | 19 | |
F151 Humanized HC1 | 20 | |
F151 Humanized HC2a | 21 | |
F151 Humanized HC2b | 22 | |
F151 Humanized HC2c | 23 | |
F151 Humanized HC3a | 24 | |
F151 Humanized HC3b | 25 | |
F151 VL | 26 | |
F151 Humanized LC1 | 27 | |
F151 Humanized LC2a | 28 | |
F151 Humanized LC2b | 29 | |
F151 Humanized LC3a | 30 | |
F151 Humanized LC3b | 31 | |
B21 HCDR3 | WEYDGYYDLDY | 32 |
B21 HCDR2 | WIDPENGDTGYARKFQG | 33 |
B21 HCDR1 | GFNIKDYYLH | 34 |
B21 LCDR3 | LQGTHFPYT | 35 |
B21 LCDR2 | LVSKLDS | 36 |
B21 LCDR1 | KSSQSLLYSNGKTYLN | 37 |
B21 VH | 38 | |
B21 VL | 39 | |
C63 HCDR3 | EDYGGDY | 40 |
C63 HCDR2 | EIRSKSNNYATHYAESVKG | 41 |
C63 HCDR1 | GFTFSNYWMN | 42 |
C63 LCDR3 | QQYYSYPYT | 43 |
C63 LCDR2 | WASTRES | 17 |
C63 LCDR1 | KSSQSLLYSSDQRNYLA | 44 |
C63 VH | 45 | |
C63 VL | 46 | |
I22 HCDR3 | FEYDGNYSPLDF | 47 |
I22 HCDR2 | WVDPENGDSDYAPKFQ | 48 |
I22 HCDR1 | GFNIKDYYMH | 49 |
I22 LCDR3 | QNDHSYPLT | 50 |
I22 LCDR2 | GASTRES | 51 |
I22 LCDR1 | KSSQSLLNSGNQKNYLA | 52 |
I22 VH | 53 | |
I22 VL | 54 | |
I54 HCDR3 | FEYDGNYSPLDF | 55 |
I54 HCDR2 | WVDPENGDSDYAPKFQG | 56 |
I54 HCDR1 | GFNIKDYYMH | 57 |
I54 LCDR3 | MQGTHFPYT | 58 |
I54 LCDR2 | LVSKLDS | 59 |
I54 LCDR1 | KSSQSLLYSNGETYLN | 60 |
154 VH | 61 | |
154 VL | 62 | |
B21/I22/I54 HCDR3 consensus | X1EYDGX2YX3X4LDX5 | 63 |
where: | ||
X1 is W or F; | ||
X2 is N or no amino acid; | ||
X3 is Y or S; | ||
X4 is D or P; and | ||
X5 is F or Y. | ||
B21/I22/I54 HCDR2 consensus | WX1DPENGDX2X3YAPKFQG | 64 |
where: | ||
X1 is I, or V; | ||
X2 is T, or S; and | ||
X3 is G, or D. | ||
B21/I22/I54 HCDR1 consensus | GFNIKDYYX1H | 65 |
where X1 is L, or M. | ||
F151/C63/I22 LCDR3 consensus | QX1X2X3SX4PX5T | 66 |
where: | ||
X1 is Q or N; | ||
X2 is Y, F, D or H; | ||
X3 is Y, F, H or W; | ||
X4 is Y, F, T or H; and | ||
X5 is W, Y, F, H or L. | ||
F151/C63/I22 LCDR2 consensus | X1ASTRX2 | 67 |
where: | ||
X1 is W or G; and | ||
X2 is E, D, Q or N | ||
F151/C63/I22 LCDR1 consensus | KSSQSLLX1X2SX3QX4NX5LA | 68 |
where: | ||
X1 is W, H, Y or F; | ||
X2 is S or G; | ||
X3 is N or D; | ||
X4 is K or R; | ||
X5 is H or Y. | ||
B21/I54 LCDR3 consensus | X1QGTHFPYT | 69 |
where: | ||
X1 is L or M; | ||
B21/I54 LCDR2 both identical | LVSKLDS | 36 |
B21/I54 LCDR1 consensus | KSSQSLLYSNGX1TYLN | 70 |
where: | ||
X1 is K or E; |
a heavy chain variable region comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in 13, 14, and 15, respectively, and one or more amino acid substitution at positions selected from the group consisting of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, and H108; and/or
a light chain variable region comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in 16, 17, and 18, respectively, and one or more amino acid substitution at positions selected from the group consisting of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and L104 (according to the Kabat numbering convention).
III. Modified Anti-Kallidin or des-Arg10-Kallidin antibodies
i) Reducing Immunogenicity
ii) Effector Functions and Fc Modifications
iii) Covalent Attachment
IV. Expression of Anti-Kallidin or des-Arg10-Kallidin Antibodies, or Antigen Binding Fragments Thereof
V. Pharmaceutical Formulations and Methods of Administration of Anti-Kallidin or des-Arg10-Kallidin Antibodies.
VI. Methods of Treating Kallidin or des-Arg10-Kallidin-Associated Disease or Disorders
VII. Exemplification
Example 1: Hybridoma Production: Immunization of Mice with Kallidin Peptide Conjugated to KLH and Antibody Generation against Human BKR1 Ligands
Peptide No. | SEQ ID NO. | Peptide Sequence | Peptide Name | Alternative Name |
1 | 5 | RPPGFSPFR | bradykinin | BK |
2 | 117 | biotin-RPPGFSPFR | b-BK | |
3 | 71 | RPPG FSPFR-biotin | BK-b | |
4 | 72 | KLH-RPPGFSPFR | KLH-BK | |
5 | 73 | RPPGFSPFR-KLH | BK-KLH | |
6 | 1 | KRPPGFSPFR | kallidin | KD |
7 | 74 | biotin-KRPPGFSPFR | b-KD | |
8 | 75 | KRPPGFSPFR-biotin | KD-b | |
9 | 76 | KLH-KRPPGFSPFR | KLH-KD | |
10 | 77 | KRPPGFSPFR-KLH | KD-KLH | |
11 | 6 | RPPGFSPF | desArg9bradykinin | DABK |
12 | 78 | biotin-RPPGFSPF | b-DABK | |
13 | 79 | RPPGFSPF-biotin | DABK-b | |
14 | 80 | KLH-RPPGFSPF | KLHDABK | |
15 | 81 | RPPGFSPF-KLH | DABK-KLH | |
16 | 2 | KRPPGFSPF | desArg10kallidin | DAKD |
17 | 82 | biotin-KRPPGFSPF | b-DAKD | |
18 | 83 | KRPPGFSPF-biotin | DAKD-b | |
19 | 84 | KLH-KRPPGFSPF | KLH-DAKD | |
20 | 85 | KRPPGFSPF-KLH | DAKD-KLH | |
21 | 3 | RRPPGFSPFR | Kallidin like peptide | KLP |
22 | 86 | biotin-RRPPGFSPFR | b-KLP | |
23 | 87 | RRPPGFSPFR-biotin | KLP-b | |
24 | 88 | KLH-RRPPGFSPFR | KLH-KLP | |
25 | 89 | RRPPGFSPFR-KLH | KLP-KLH | |
26 | 90 | RRPPGFSPF | desArg10kallidin like peptide | DAKLP |
27 | 91 | biotin-RRPPGFSPF | b-DAKLP | |
28 | 92 | RRPPGFSPF-biotin | DAKLP-b | |
29 | 93 | KLH-RRPPGFSPF | KLH-DAKLP | |
30 | 94 | RRPPGFSPF-KLH | DAKLP-b | |
31 | 95 | RPPGF | bradykinin1-5 | BK15 |
32 | 96 | biotin-RPPGF | b-BK15 |
Example 2: Characterization and Selection of Hybridomas Expressing Antibodies Against Human BKR1 Ligands
DAKD-b | KD-b | |||
Antibody | koff | KD | koff | KD |
C63 | 9.36E-05 | 1.42E-10 | 1.00E-04 | 2.21E-10 |
B21 | 9.89E-05 | 4.15E-11 | 2.04E-04 | 8.40E-11 |
F151 | 1.36E-04 | 1.62E-10 | 2.00E-05 | 2.88E-11 |
I22 | 3.19E-04 | 2.17E-10 | 2.10E-05 | 4.40E-12 |
I54 | 3.06E-05 | 9.53E-12 | 3.88E-05 | 1.12E-11 |
DAKLP-b | KLP-b | |||
koff | KD | koff | KD | |
C63 | n/b | n/b | n/b | n/b |
B21 | 2.30E-04 | 1.34E-10 | 1.12E-04 | 1.92E-10 |
F151 | 6.58E-05 | 2.12E-10 | ≤1.0E-06 | ≈1.66E-11 |
I22 | ≤1.0E-06 | ≈1.83E-12 | 1.03E-05 | 1.82E-12 |
I54 | 5.66E-05 | 1.17E-11 | 6.04E-05 | 9.56E-12 |
n/b = no binding |
EXAMPLE 3: Generation of Surrogate Antibody for Murine Animal Studies
Clone | b-3G-DABK | b-3G-DAKD | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | 1.5E+06 | 2.3E-04 | 1.6E-10 | 4.7E+05 | 4.0E-04 | 8.8E-10 |
UR11 | 2.0E+05 | 3.0E-04 | 1.5E-09 | 3.0E+05 | 1.6E-03 | 5.2E-09 |
DD7 | 2.3E+05 | 6.0E-04 | 2.7E-09 | 2.1E+05 | 1.4E-03 | 6.6E-09 |
EE1 | 4.4E+05 | 1.2E-04 | 2.8E-10 | 4.4E+05 | 2.0E-04 | 4.5E-10 |
EE36 | 4.3E+03 | 5.3E-04 | 1.2E-07 | n/b | n/b | n/b |
UR29 | n/b* | n/b | n/b | n/b | n/b | n/b |
JK3 | 3.44E+05 | 3.91E-05 | 1.14E-10 | 3.18E+05 | 5.07E-05 | 1.60E-10 |
LR4 | n/b | n/b | n/b | n/b | n/b | n/b |
LR16 | n/b | n/b | n/b | n/b | n/b | n/b |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | n/b | n/b | n/b |
NR15 | n/b | n/b | n/b | n/b | n/b | n/b |
n/b* = non-specific binding, n/b = no binding |
Clone | b-3G-DAKLP | b-3G-BK | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | 3.1E+05 | 6.1E-04 | 2.0E-09 | 3.0E+05 | 7.1E-04 | 2.3E-09 |
UR11 | n/b | n/b | n/b | n/b | n/b | n/b |
DD7 | 1.7E+05 | 2.1E-03 | 1.2E-08 | 1.3E+05 | 8.8E-04 | 6.8E-09 |
EE1 | 4.2E+05 | 2.9E-04 | 6.8E-10 | 2.5E+05 | 2.6E-03 | 1.1E-08 |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | n/b | n/b | n/b | n/b | n/b | n/b |
JK3 | ND | ND | ND | n/b | n/b | n/b |
LR4 | n/b | n/b | n/b | n/b | n/b | n/b |
LR16 | n/b | n/b | n/b | n/b | n/b | n/b |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | n/b | n/b | n/b |
NR15 | n/b | n/b | n/b | n/b | n/b | n/b |
n/b = no binding; ND = not determined |
Clone | b-3G-KLP | b-3G-KD | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | 3.8E+05 | 5.7E-04 | 1.5E-09 | n/b | n/b | n/b |
UR11 | n/b | n/b | n/b | 1.2E+05 | 1.3E-03 | 1.1E-08 |
DD7 | 1.5E+05 | 2.1E-03 | 1.5E-08 | 2.2E+05 | 1.8E-03 | 8.4E-09 |
EE1 | 4.0E+05 | 2.1E-03 | 5.3E-09 | n/b | n/b | n/b |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | n/b | n/b | n/b | n/b | n/b | n/b |
JK3 | ND | ND | ND | n/b | n/b | n/b |
LR4 | n/b | n/b | n/b | n/b | n/b | n/b |
LR16 | n/b | n/b | n/b | n/b | n/b | n/b |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | n/b | n/b | n/b |
NR15 | n/b | n/b | n/b | n/b | n/b | n/b |
n/b = no binding; ND = not determined |
Clone | DABK-b | DAKLP-b | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | n/b | n/b | n/b | n/b | n/b | n/b |
UR11 | n/b | n/b | n/b | n/b | n/b | n/b |
DD7 | n/b | n/b | n/b | n/b | n/b | n/b |
EE1 | n/b | n/b | n/b | n/b | n/b | n/b |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | 1.5E+06 | 5.8E-05 | 3.9E-11 | 3.0E+06 | 2.1E-03 | 6.8E-10 |
JK3 | n/b | n/b | n/b | n/b | n/b | n/b |
LR4 | n/b | n/b | n/b | n/b | n/b | n/b |
LR16 | n/b | n/b | n/b | n/b | n/b | n/b |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | n/b | n/b | n/b |
NR15 | n/b | n/b | n/b | n/b | n/b | n/b |
n/b = no binding; ND = not determined |
Clone | BK-b | b-BK | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | n/b | n/b | n/b | n/b | n/b | n/b |
UR11 | n/b | n/b | n/b | n/b | n/b | n/b |
DD7 | n/b | n/b | n/b | n/b | n/b | n/b |
EE1 | n/b | n/b | n/b | n/b | n/b | n/b |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | 1.5E+06 | 1.0E-04 | 7.2E-11 | n/b | n/b | n/b |
JK3 | n/b | n/b | n/b | n/b | n/b | n/b |
LR4 | n/b | n/b | n/b | n/b | n/b | n/b |
LR16 | n/b | n/b | n/b | n/b | n/b | n/b |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | 8.69E+04 | 8.77E-04 | 1.01E-08 |
NR15 | n/b | n/b | n/b | 2.95E+05 | 1.09E-03 | 3.68E-09 |
n/b = no binding; ND = not determined |
Clone | b-DABK | b-DAKD | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | n/b | n/b | n/b | n/b | n/b | n/b |
UR11 | n/b | n/b | n/b | n/b | n/b | n/b |
DD7 | n/b | n/b | n/b | n/b | n/b | n/b |
EE1 | n/b | n/b | n/b | n/b | n/b | n/b |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | n/b | n/b | n/b | n/b | n/b | n/b |
JK3 | n/b | n/b | n/b | n/b | n/b | n/b |
LR4 | 1.48E+05 | 1.04E-03 | 7.15E-09 | 3.27E+05 | 7.63E-04 | 2.36E-09 |
LR16 | 4.34E+05 | 4.38E-05 | 1.01E-10 | 2.07E+05 | 3.39E-03 | 1.65E-08 |
LR6 | n/b | n/b | n/b | n/b | n/b | n/b |
LR12 | 2.91E+05 | 5.40E-04 | 3.63E-09 | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | n/b | n/b | n/b |
NR15 | n/b | n/b | n/b | n/b | n/b | n/b |
n/b = no binding; ND = not determined |
Clone | b-DAKLP | b-KD | ||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DD20 | n/b | n/b | n/b | n/b | n/b | n/b |
UR11 | n/b | n/b | n/b | n/b | n/b | n/b |
DD7 | n/b | n/b | n/b | n/b | n/b | n/b |
EE1 | n/b | n/b | n/b | n/b | n/b | n/b |
EE36 | n/b | n/b | n/b | n/b | n/b | n/b |
UR29 | n/b | n/b | n/b | n/b | n/b | n/b |
JK3 | n/b | n/b | n/b | n/b | n/b | n/b |
LR4 | 1.84E+05 | 2.58E-04 | 1.40E-09 | n/b | n/b | n/b |
LR16 | 2.34E+05 | 1.11E-04 | 4.74E-10 | n/b | n/b | n/b |
LR6 | 6.80E+05 | 4.01E-04 | 7.45E-10 | n/b | n/b | n/b |
LR12 | n/b | n/b | n/b | n/b | n/b | n/b |
NR1 | n/b | n/b | n/b | 1.66E+05 | 5.81E-03 | 3.56E-08 |
NR15 | n/b | n/b | n/b | 7.66E+05 | 5.66E-03 | 7.41E-09 |
n/b = no binding; ND = not determined |
Antibody Families | Immunogens | Representative antibodies | Binding Specificity |
Family 1 | KD-KLH + KLH-KD or DAKD-KLH + KLH-DAKD | F151, B21, I22, I54 | N-terminus of DAKD (DAKLP) and KD (KLP) |
Family 2 | KD-KLH + KLH-KD or DAKD-KLH + KLH - DAKD | C63 | N-terminus of DAKD and KD |
Family 3 | KLH-3G-DABK | EE1, DD20, JK3 | C-terminus of DABK and DAKD (DAKLP) |
Family 4 | KLH-BK and BSA-BK | NR15, NR1 | C-terminus of BK and KD |
Family 5 | KLH-BK | UR29 | N-terminus of BK and DABK |
Family 6 | KLH-BK | UR11 | BK, DABK and DAKD |
Family 7 | KLH-DABK or KLH-DAKD | LR4, LR6, LR12 and LR16 | no binding with native peptides |
Example 4: Characterization of des-arg-Kinin Ligand Depletion using Calcium Mobilization
Depletion of DABK | Depletion of DAKD | Depletion of DAKLP | ||||||||
Family | Antibody | DABK (Mean Molar Ratio) | SD Molar Ratio | n | DAKD (Mean Molar Ratio) | SD Molar Ratio | n | DAKLP (Mean Molar Ratio) | SD Molar Ratio | n |
1 | F151 | IA100 | 5 | 0.08 | 0.04 | 7 | 0.15 | 0.04 | 4 | |
1 | B21 | IA100 | 1 | 0.15 | 0.04 | 3 | 0.67 | 1 | ||
1 | I22 | IA100 | 1 | 0.07 | 0.02 | 3 | 0.21 | 1 | ||
1 | 154 | IA100 | 1 | 0.15 | 0.05 | 3 | 0.35 | 1 | ||
2 | C63 | IA100 | 1 | 0.08 | 0.02 | 3 | 5.85 | 1 | ||
3 | EE1 | 1.03 | 0.52 | 5 | 0.86 | 0.52 | 3 | 0.57 | 0.36 | 4 |
3 | DD20 | 3.45 | 1.34 | 3 | 1.82 | 0.76 | 3 | 1.31 | 0.86 | 3 |
DD7 | 2.18 | 0.45 | 3 | 4.22 | 0.95 | 3 | 5.34 | 1.22 | 2 | |
3 | JK3 | 1.86 | 0.03 | 2 | ND | 1.44 | 0.03 | 2 | ||
4 | MBK3 | ND | ND | ND | ||||||
4 | NR15 | ND | ND | ND | ||||||
4 | NR1 | ND | ND | ND | ||||||
5 | UR29 | 0.60 | 0.12 | 5 | IA200 | 3 | IA300 | 4 | ||
6 | UR11 | 6.99 | 1.61 | 3 | 19.65 | 14.95 | 3 | 11.09 | 3.13 | 2 |
7 | LR4 | IA100 | 1 | IA400 | 1 | IA400 | 1 | |||
7 | LR6 | IA100 | 1 | IA100 | 1 | ND | ||||
7 | LR12 | IA100 | 1 | IA100 | 1 | ND | ||||
7 | LR16 | IA100 | 1 | IA100 | 1 | ND | ||||
Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B1 Receptor. The antibody-ligand mixture was added to HEK mBKR1 cells pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluorescence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fit in Graph Pad Prism V4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concentration of ligand that was used for the various experiments. | ||||||||||
Molar Ratio for ligand depletion = [IC50 of Antibody]/ [Ligand] | ||||||||||
SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100nM; IA200 = Inactive at 200nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400nM |
Depletion of BK | Depletion of KD | Depletion of KLP | ||||||||
Family | Antibody | BK (Mean Molar Ratio) | SD Molar Ratio | n | KD (Mean Molar Ratio) | SD Molar Ratio | n | KLP (Mean Molar Ratio) | SD Molar Ratio | n |
0.02 | ||||||||||
1 | F151 | IA100 | 5 | 0.14 | 0.05 | 5 | 0.15 | 3 | ||
1 | B21 | IA100 | 1 | 0.33 | 1 | ND | ||||
1 | I22 | IA100 | 1 | 0.22 | 1 | ND | ||||
1 | 154 | IA100 | 1 | 0.30 | 1 | ND | ||||
2 | C63 | IA100 | 1 | 0.23 | 1 | ND | ||||
3 | EE1 | IA300 | 4 | IA300 | 4 | IA150 | 1 | |||
3 | DD20 | IA600 | 4 | IA600 | 5 | IA150 | 1 | |||
DD7 | 7.11 | 3.62 | 3 | 17.37 | 12.11 | 3 | 4.27 | 1 | ||
3 | JK3 | IA300 | 2 | IA300 | 2 | ND | ||||
4 | MBK3 | 22.11 | 14.10 | 9 | 3.46 | 2.64 | 6 | 9.45 | 1 | |
4 | NR15 | 15.26 | 11.51 | 5 | 4.34 | 2.55 | 5 | 11.18 | 1 | |
4 | NR1 | 39.31 | 1 | 42.15 | 1 | 32.58 | 1 | |||
5 | UR29 | 1.15 | 0.86 | 5 | 0.30 | 0.08 | 2 | 0.41 | 1 | |
6 | UR11 | 5.41 | 0.80 | 2 | 25.21 | 4.54 | 2 | 1.53 | 1 | |
7 | LR4 | IA100 | 1 | IA100 | 1 | ND | ||||
7 | LR6 | IA100 | 1 | IA100 | 1 | ND | ||||
7 | LR12 | IA100 | 1 | IA100 | 1 | ND | ||||
7 | LR16 | IA100 | 1 | IA100 | 1 | ND | ||||
Antibodies were pre-incubated with a set concentration of ligand, usually an EC70-80 for activating calcium mobilization at the Bradykinin B2 Receptor. The antibody-ligand mixture was added to MRC5 Fetal Lung Fibroblasts (ATCC CCL-171) pre-loaded with a calcium sensing dye (Fluo-4AM or Fluo-8AM) on the Hamamatsu FDSS6000 instrument, and calcium mobilization was monitored. Data was exported as a max-min relative fluorescence of the biological response, and IC50 for ligand depletion was calculated using sigmoidal curve fit in Graph Pad Prism V4.03. Data reported as molar ratio for ligand depletion by the antibody to standardize the different concentration of ligand that was used for the various experiments. | ||||||||||
Molar Ratio for ligand depletion = [IC50 of Antibody]/[Ligand] | ||||||||||
SD = Standard Deviation; ND = not determined; IA100 = Inactive at 100nM; IA150 = Inactive at 150nM; IA300 = Inactive at 300nM; IA400 = Inactive at 400nM; IA600 = Inactive at 600nM |
Example 5: Engineering of F151: Humanization, Stabilization and Mutation of Unwanted Sequence Motifs
1. HUMANIZATION
2. STABILIZATION
a) KNOWLEDGE-BASED APPROACH
b) 3D AND MD-BASED APPROACHES
3. HUMANIZATION BY GRAFTING
4. MUTATION OF UNWANTED SEQUENCE MOTIFS
5. ORIGINAL SEQUENCES OF MURINE F151 VARIABLE DOMAINS
6. ENGINEERED SEQUENCES
a) Background
Light Chain | Light Chain | (LC1) Humanizing mutations | (LC2a) Humanizing + stabilizing mutations | (LC2b) Humanizing + stabilizing mutations + anti-aggregation mutations | (LC3a) Grafting CDRs + Vernier residues | (LC3b) Grafting CDRs only |
Sequential numbering | Kabat Numbering | |||||
Ser5 | Ser5 | Thr | Thr | Thr | Thr | |
Ser9 | Ser9 | Asp | Asp | |||
Ala12 | Ala12 | Ser | Ser | |||
Val13 | Val13 | Ala | Ala | Ala | ||
Val15 | Val15 | Leu | Leu | |||
Glu17 | Glu17 | Asp | Asp | Asp | ||
Lys18 | Lys18 | Arg | Arg | Arg | Arg | Arg |
Val19 | Val19 | Ala | Ala | |||
Met21 | Met21 | Ile | Ile | Ile | Ile | |
Ser22 | Ser22 | Asn | Asn | |||
Gln48 | Gln42 | Lys | Lys | Lys | ||
Ser49 | Ser43 | Pro | Pro | |||
Pro52 | Pro46 | Leu | ||||
Thr69 | Thr63 | Ser | Ser | Ser | Ser | |
Val84 | Val78 | Leu | Leu | |||
Lys85 | Lys79 | Gln | Gln | Gln | Gln | Gln |
Leu89 | Leu83 | Lys | Val | Val | ||
Ile91 | Ile85 | Thr | Thr | Val | Val | |
Gly106 | Gly100 | Gln | Gln | |||
Leu110 | Leu104 | Val | Val | |||
Mutations: | 5 | 10 | 11 | 15 | 16 |
a) Engineered light chain sequences:
LC1 (SEQ ID NO:27), humanizing mutations are underlined, CDRs and vernier zones are
in bold:
LC2a (SEQ ID NO:28), humanizing mutations are underlined, CDRs and vernier zones are
in bold, stabilization mutations are in italics (T at position 5, S at position 12,
I at position 21, S at position 69, T at position 91 shown below) :
LC2b (SEQ ID NO:29) humanizing mutations are underlined, CDRs and vernier zones are
in bold, stabilization mutations are in italics (T at position 5, S at position 12,
I at position 21, S at position 69, T at position 91 shown below) and an anti-aggregation
mutation is K at position 89:
LC3a (SEQ ID NO:30), grafted mutations shown in underline and CDRs and vernier zones
shown in bold:
LC3b (SEQ ID NO:31), grafted mutations shown in underline and CDRs and vernier zones
shown in bold:
Note that L at position 52 is a vernier residue that is mutated to human.
c) Engineered Heavy chain sequences
HC1 (SEQ ID NO:20), humanizing mutations are underlined, CDRs and vernier zones are
in bold:
HC2a (SEQ ID NO:21), humanizing mutations are underlined, CDRs and vernier zones are
in bold, stabilization mutations are in italics (Q at position 1, A at position 9,
G at position 44, Y at position 80 and E at position 90 shown below):
HC2b (SEQ ID NO:22), humanizing mutations are underlined, CDRs and vernier zones are
in bold, stabilization mutations are in italics (Q at position 1, A at position 9,
G at position 44, E at position 62, Y at position 80 and E at position 90 shown below):
No human epitopes were identified for sequence HC2b in IEDB database.
HC2c (SEQ ID NO:23), humanizing mutations are underlined, CDRs and vernier zones are
in bold, stabilization mutations are in italics (Q at position 1, A at position 9,
G at position 44, Y at position 80 and E at position 90 shown below) and an anti-aggregation
mutation at K at position 86:
HC3a (SEQ ID NO:24), grafted mutations shown in underline and CDRs and vernier zones
shown in bold:
LC3b (SEQ ID NO:25), grafted mutations shown in underline and CDRs and vernier zones
shown in bold:
Note that the following Vernier Residue are mutated to human: V at position 2, M at
position 48, V at position 68, M at position 70 and T at position 74.
No human epitopes were identified for sequence HC3b in IEDB database.
HC3b germinality index = 83% with Z12316_1_V_J00235_1_D_U42590_1_J [1-18/DP-14],
Residue | Proposed Change | Calculated Gth | Accept Change |
Ser-5 | Thr | 2.32286 | Yes |
Ala-12 | Ser | 0.75228 | Yes |
Met-21 | Ile | 0.768959 | Yes |
Pro-52 | Leu | 1.70059 | No - Vernier region |
Thr-69 | Ser | 1.10843 | Yes |
Lys-86 | Glu | 2.00115 | No - changed to Gln during humanization |
Ile-91 | Thr | 1.27255 | Yes |
Residue | Proposed Change | Calculated Gth | Accept Change |
Glu-1 | Gln | 0.562423 | Yes |
Ile-2 | Val | 2.15882 | No - Vernier region |
Pro-9 | Ala | 0.505324 | Yes |
Thr-16 | Ala | 1.50552 | Already changed to Ala in humanization |
Val-20 | Leu | 2.21586 | No - not in germline sequence |
Ser-40 | Arg | 1.03643 | No - not in germline sequence |
His-41 | Pro | 1.67738 | Already changed to Pro in humanization |
Ser-44 | Gly | 1.5068 | Yes |
Gln-62 | Glu | 0.74934 | No - not in germline sequence |
Arg-65 | Lys | 2.32314 | No - not in germline sequence |
Phe-80 | Tyr | 1.30935 | Yes |
His-82 | Gln | 2.24674 | No - not in germline sequence |
Asp-89 | Glu | 1.65409 | Already changed to Glu in humanization |
Asn-98 | Arg | 3.65643 | No - Vernier region |
Combination* | Additional changes suggested | Accept Change |
L1 (46->P & 48->Q) | K48->Q | No - K48 humanizing mutation |
L2 (51->K) | None - already K51 | None |
L3 (80->T) | None - already T80 | None |
L4 (82->S) | None - already S82 | None |
L5 (90->A, 91->T) | None - already A90, T91 suggested above (Table 1) | None |
H1 (15->G) | None - already G15 | None |
H2 (62->E, 63->K, 64->F) | Q62->E, already K63 and F64 | Yes - considered in HC2b |
H3 (87->T, 88->S, 89->D) | D89->E, already T87 and S88 | Yes - potential salt bridges with K63 and K43 |
S1 (L1 & L5) | K48->Q | No - K48 humanizing mutation |
S2 (H1 & H3) | D89->E | No (see H3) |
*Note: Sequential numbering used to refer to residues |
Light Chain Residue* | Additional changes suggested | Accept Change |
15->L | V15->L | No - V15 in Vk1 germline |
96->Q | None - already Q96 | None |
38->Y | None - already Y38 | None |
112->I | None - already 1112 | None |
69->S | G69->S | No - G69 is in Vernier Region |
21->I | M21->I | Already changed (see Table 19) |
*Note: Sequential numbering used to refer to residues |
Example 6: Characterization of Humanization Variants
HC1/LC1 | HC2a/LC2a | |||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DAKD-b | 4.16E+05 | 6.00E-06 | 1.45E-11 | 6.66E+05 | 1.22E-05 | 1.83E-11 |
KD-b | 4.24E+05 | 1.74E-07 | 3.94E-13 | 7.03E+05 | 6.12E-06 | 8.71E-12 |
DAKLP-b | 5.00E+05 | 7.96E-06 | 1.60E-11 | 4.10E+05 | 5.67E-06 | 1.38E-11 |
KLP-b | 4.81E+05 | 2.67E-06 | 5.54E-12 | 6.15E+05 | 2.68E-05 | 4.34E-11 |
HC2b/LC2a | HC2c/LC2b | |||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DAKD-b | 4.17E+05 | 1.05E-05 | 2.57E-11 | 4.81E+05 | 4.34E-05 | 9.01E-11 |
KD-b | 3.75E+05 | 1.66E-06 | 4.72E-12 | 5.64E+05 | 9.08E-06 | 1.74E-11 |
DAKLP-b | 4.46E+05 | 1.30E-05 | 2.97E-11 | 9.03E+05 | 1.10E-05 | 1.21E-11 |
KLP-b | 4.01E+05 | 2.20E-06 | 5.76E-12 | 5.16E+05 | 1.02E-05 | 1.98E-11 |
HC3a/LC3a | HC3b/LC3b | |||||
Ka(1/Ms) | Kd(1/s) | KD(M) | Ka(1/Ms) | Kd(1/s) | KD(M) | |
DAKD-b | 5.06E+05 | 1.28E-05 | 2.53E-11 | 3.85E+05 | 5.15E-05 | 1.35E-10 |
KD-b | 4.27E+05 | 2.95E-06 | 6.78E-12 | 2.51 E+05 | 3.02E-06 | 1.44E-11 |
DAKLP-b | 4.65E+05 | 1.42E-05 | 3.05E-11 | 7.04E+04 | 2.76E-03 | 4.05E-08 |
KLP-b | 5.02E+05 | 5.43E-07 | 1.06E-12 | 5.39E+05 | 2.72E-04 | 5.26E-10 |
For comparison: Ka(1/Ms) of mF151 was 7.84E+05 for DAKD-b, 8.30E+05 for KD-b, 1.81E+06 for DAKLP-b, and 1.12E+06 for KLP-b |
Thermostability | Stability | Intactness Confirmation (LC, HC) | N-terminal sequence confirmation | |||
Variant | 1D-gel | SEC | Biacore | SEC | LC-MS | N-terminal sequencing |
HC1/LC1 | No Ag/Deg | No Ag/Deg | 45C slightly faster off rate than 4C | No Ag/Deg | LC(+1 Da off) HC(+1 Da off) G0 dominant | N terminal of LC & HC intact |
HC2a/LC2a | No Ag/Deg | No Ag/Deg | 45C slightly faster off rate than 4C | No Ag/Deg | LC(spot on) HC(spot on) G0 dominant | N terminal of LC & HC intact |
HC2b/LC2a | No Ag/Deg | No Ag/Deg | 45C slightly faster off rate than 4C | No Ag/Deg | LC(+1 Da off) HC(+1 Da off) G0 dominant | N terminal of LC & HC intact |
HC2c/LC2b | X | X | X | X | X | X |
HC3a/LC3a | No Ag/Deg | No Ag/Deg | 45C slightly faster off rate than 4C | No Ag/Deg | LC(-2Da off) HC(-2Da off) G0 dominant | N terminal of LC & HC intact |
HC3b/LC3b | X | X | X | X | X | X |
Thermostability = Incubation at 4°C (control) and 45°C for 3 days; 1 D-gel was under nonreducing conditions; Stability = 2 cycles of freeze/thaw; LC-MS = reduced and reduced/deglycosylation | ||||||
Abbreviations: Ag=aggregation, Deg=degradation, X = no data presented |
Example 7: Crystal Structure of Humanized Antibody F151 against BRK1 Ligand Kallidan and des-arg10-Kallidin
Peptide name | SEQ ID NO: | Sequence |
1 2 3 4 5 6 7 8 9 10 | ||
KD (Kallidin) | 1 | Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg |
DAKD (des-Arg10-Kallidin) | 2 | Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe |
KLP (rodent KD ortholog) | 3 | Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg |
BK (Bradykinin) | 5 | Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg |
Residue | Role in KD Binding or CDR Stabilization | Functional Substitution |
Tyr-L31 | •Edge-on hydrophobic interactions with Pro4 | •His to add an H-bond with amide N of Pro5 |
•Along with Tyr-L38 and Tyr-L98 form three orthogonal planes that surround the 90deg turn of KD at Pro4 | •other aromatic a.a, such as Trp, and Phe | |
Tyr-L38 | •hydrophobic stacking with Pro4 | •His to add an H-bond with carbonyl O of Arg2 |
•Along with Tyr-L31 and Tyr-L98 form three orthogonal planes that surround the 90deg turn of KD at Pro4 | ||
Tyr-L55 | •cation- interaction with amidinium ion of Lys1 sidechain | •Trp to pair with H105 mutations of Gln, Asn, Glu or Asp (maintaining H-bond); Other variants of His_H105 are Tyr and Ser. |
•H-bond with His-H105; Tyr-L55--His-H105 pairing adds stabilization between L2 and H3 loops | •Other aromatic a.a., such as Trp, His and Phe | |
Glu-L61 | •Forming key salt bridges with Lys1 sidechain | •Asp, Gln, ASN (Asp is seen in B21 and I54 already, Figure 7) |
Tyr-L97 | •H-bond with amide N of Trp-L56 | •Aromatic a.a. such as Phe or His (too tight space for Trp) |
•Forming pocket for Arg1 extended sidechain | ||
Tyr-L98 | •Along with Tyr-L31 and Tyr-L38 form three orthogonal planes that surround the 90deg turn of KD at Pro4 | •Other aromatic a.a, such as Phe, Trp or His |
Tyr-L100 | •Forming pocket surface for Pro3 | •Aromatic a.a., such as Phe (better hydrophobic interactions with Pro3) |
• Part of the aromatic a.a. cluster interface between L/H chains, further including Tyr-L42, Tyr-L93, Trp_L102, Phe-L104 and Tyr-H35, Trp-H47, Tyr-H50, Tyr-H99, Trp-H110 | • Other variants seen are Thr (in B21 and I54) and His (in I22) | |
• Partial stacking with Tyr-H50 | ||
Trp-L102 | • Part of the aromatic a.a. cluster interface between L/H chains | • Other aromatic a.a, such as Tyr (in C63 and B21), Phe and His |
• Stacking with Trp-H47 | • Other hydrophobic residue, such as L (in I22) | |
Asn-H33 | • Close to Phe6 sidechain but incompatible in polarity, no other roles seen either; can be a target for affinity maturation | • Replace with aromatic/hydrophobic a.a., such as Trp (seen in C63) or Tyr (seen inB21, I22, I54) |
Asp-H52 | • Salt bridge with Arg-H101, stabilizing H1 and H3 loops | • Mutate as a pair with Arg-H101 to reversely charged a.a., such Arg-H52/Asp-H101, or a pair of hydrophobic a.a. (Leu, Ile, Val, Met, Phe, Tyr, Trp, Ala) to form a cluster with Phe6 of KD |
Tyr-H54 | • Close to Pro8 but no specific interactions | • Mutated to negatively charged a.a. to stabilize Arg-H101, such D or E (seen in B21, I22 and I54) or N or Q, also provide H-bond with carbonyl O of Pro8 (A Lys in C63, which can be charge-reversed to Glu) |
• Close to Arg-H101, but no charge interactions | ||
Tyr-H99 | • Part of aromatic interface between H and L chains | • Mutate to small aromatic a.a. except W, such as Phe and His |
• H-bond with Asn-H33 | ||
• Tight space | ||
Arg-H101 | • H-bond with amide of Pro8 | • Mutate as a pair with Asp-H52 to reversely charged a.a., such Arg-H52/Asp-H101, or a pair of hydrophobic a.a. to form a cluster with Phe6 of KD |
• supported by Asp-H52 (salt bridge) | ||
Tyr-H102 | • Half-way intercalating into Phe9 and Pro8, hydrophobic interactions with KD | • Phe can be better, Trp or His may be OK too |
Asp-H104 | • Key residue to salt-bridge with Arg2 | • Glu to maintain salt bridge with Arg2 |
• Bigger a.a to fill the gap from Pro3, such as Tyr as seen in B21, I22 and I54 | ||
Asp-H108 | • Key residue to salt bridge with N-term -NH3+ of KD | • Glu |
• H-bond with Tyr-H33, stabilizing H3 loop | ||
• Conserved residue! Not in CDR |
Example 8: In vivo Pharmacology of anti-BKR1-Ligand Antibodies in Pain Models
Animals
A. Formalin-induced acute inflammatory pain
Group | Dose (mg/kg, i.v.) | A.U.C. ±SEM (15-45 min) | Reversal of pain-like behavior (in %) ±SEM (15-45 min) |
Isotype-control 1B7.11 (EE1) | 30 | 63.6±2.9 | 0±5 |
EE1 | 2.5 | 41.6±3.4 (***) | 35±5 |
10 | 42.9±2.9 (***) | 33±5 | |
30 | 36.5±4.3 (***) | 45±7 | |
Isotype-control 1B7.11 (F151) | 10 | 57.3±3 | 0±5 |
F151 | 2.5 | 48.9±3.8 (NS) | 15±7 |
10 | 45.0±2.8 (*) | 21±5 | |
*, p<0.05; ***, p<0.001: Student's t-test versus adequate control was used. NS: non significant |
B. CFA (Complete Freund's adjuvant)-induced chronic inflammatory pain
B1. Mechanical Hypersensitivity
Group | Dose (mg/kg, i.v.) | Day 1 post-CFA FR (%) % effect | Day 4 post-CFA FR (%) % effect | Day 7 post-CFA FR (%) % effect | |||
Naive | n.a. | 41.3±4.4 | 100±11 | 43.8±2.6 | 100±8 | 45±5 | 100±13 |
Isotype-control 1B7.11 | 30 | 81.3±4.4 | 0±11 | 78.8±3 | 0±8 | 82.5±2.5 | 0±7 |
EE1 | 2.5 | 65±3.3 (**) | 41±8 | 66.3±3.2 (*) | 36±9 | 72.5±3.7 (*) | 27±10 |
30 | 72.5±3.1 (*) | 22±8 | 67.5±3.1 (*) | 32±9 | 63.8±3.2 (***) | 50±9 | |
FR: Frequency of Response (in %) ± SEM, % effect ± SEM, n.a. not applicable | |||||||
*, p<0.05, **, p<0.01 and ***, p<0.001, Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time |
B2. Thermal Hypersensitivity
Group | Dose (mg/kg, i.v.) | Day 1 post-CFA PWL (sec) % effect | Day 4 post-CFA PWL (sec) % effect | Day 7 post-CFA PWL (sec) % effect | |||
Isotype-control 1B7.11 | 30 | 3.3±0.3 | n.a. | 4.1±0.1 | n.a. | 3.6±0.3 | n.a. |
EE1 | 2.5 | 3.1±0.3 | -7±10 | 5.2±0.4 | 41±15 | 5.0±0.3 (*) | 46±10 |
30 | 2.6±0.2 | -20±5 | 5.7±0.6 (*) | 58±21 | 5.2±0.6 (*) | 52±17 | |
PWL: Paw withdrawal latency ± SEM, % effect ± SEM, n.a. not applicable | |||||||
*, p<0.05, Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time |
C. CCI (Chronic Constriction Injury)-induced neuropathic-like pain (Bennett's model)
C1. Mechanical Hypersensitivity
Group | Dose (mg/kg, i.v.) | Day 12 post-CCI % effect ± SEM | Day 14 post-CCI % effect ± SEM | Day 18 post-CCI % effect ± SEM |
Isotype-control 1B7.11 | 30 | 0.2±3.0 | 1.8±4.3 | 18.1±6.6 |
EE1 | 2.5 | 15.2±4.9 | 26.8±5.7 (**) | 30.3±7.1 |
30 | 15.2±5.7 | 25.7±4.5 (*) | 20.8±5.9 | |
*, p<0.05, and **,p<0.01 Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time (statistics performed on Delta ipsi values) |
C2. Thermal Hypersensitivity
Kinetic evaluation | |||||||
Group | Dose (mg/kg, i.v.) | Day 12 post-CCI PWL (sec) % effect | Day 14 post-CCI PWL (sec) % effect | Day 18 post-CCI PWL (sec) % effect | |||
naive | n.a. | 6.3±0.5 | 100±19 | 6.2±0.4 | 100±14 | 6.1±0.5 | 100±17 |
Isotype-control 1B7.11 | 30 | 3.8±0.2 | 0±7 | 3.4±0.2 | 0±5 | 3.4±0.2 | 0±7 |
EE1 | 2.5 | 4.8±0.4 | 41±16 | 4.8±0.5 | 51±16 | 5.5±0.5 (*) | 78±19 |
30 | 5.2±0.6 | 56±24 | 6.1±1.3(**) | 98±48 | 5.7±0.6 (**) | 84±22 | |
PWL: Paw withdrawal latency ± SEM, % effect ± SEM, n.a. non applicable | |||||||
*, p<0.05, and **, p<0.01 Two-Way ANOVA with time as repeated measure followed by Dunnett's test for factor group for each level of factor time |
SEQUENCE LISTING
<110> SANOFI
<120> ANTIBODIES TO BRADYKININ B1 RECEPTOR LIGANDS
<130> BET17M2471
<150> 61/616,845
<151> 2012-03-28
<150> FR 1350953
<151> 2013-02-04
<160> 137
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Homo sapiens
<400> 1
<210> 2
<211> 9
<212> PRT
<213> Homo sapiens
<400> 2
<210> 3
<211> 10
<212> PRT
<213> Mus musculus
<400> 3
<210> 4
<211> 9
<212> PRT
<213> Mus musculus
<400> 4
<210> 5
<211> 9
<212> PRT
<213> Unknown
<220>
<221> source
<223> /note="Description of Unknown: Bradykinin peptide"
<400> 5
<210> 6
<211> 8
<212> PRT
<213> Unknown
<220>
<221> source
<223> /note="Description of Unknown: Des-Arg9-Bradykinin peptide"
<400> 6
<210> 7
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (1)..(1)
<223> /replace="Phe" or "His"
<220>
<221> misc_feature
<222> (1)..(1)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (3)..(3)
<223> /replace="Asp" or "Ala" or "Val" or "Leu" or "Ile" or "Met" or "Phe" or "Tyr"
or "Trp"
<220>
<221> VARIANT
<222> (4)..(4)
<223> /replace="Phe" or "Trp" or "His"
<220>
<221> misc_feature
<222> (3)..(4)
<223> /note="Residues given in the sequence have no preference with respect to those
in the annotations for said positions"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace="Glu" or "Tyr"
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (10)..(10)
<223> /replace="Glu"
<220>
<221> misc_feature
<222> (10)..(10)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 7
<210> 8
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (3)..(3)
<223> /replace="Arg" or "Ala" or "Val" or "Leu" or "Ile" or "Met" or "Phe" or "Tyr"
or "Trp"
<220>
<221> misc_feature
<222> (3)..(3)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (5)..(5)
<223> /replace="Asp" or "Glu" or "Asn" or "Gln"
<220>
<221> misc_feature
<222> (5)..(5)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 8
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="Trp" or "Tyr"
<220>
<221> misc_feature
<222> (8)..(8)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 9
<210> 10
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (3)..(3)
<223> /replace="Phe" or "His"
<220>
<221> VARIANT
<222> (4)..(4)
<223> /replace="Phe" or "His" or "Trp"
<220>
<221> misc_feature
<222> (3)..(4)
<223> /note="Residues given in the sequence have no preference with respect to those
in the annotations for said positions"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace="Phe" or "Thr" or "His"
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="Tyr" or "Phe" or "His" or "Leu"
<220>
<221> misc_feature
<222> (8)..(8)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 10
<210> 11
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace="Asp" or "Gln" or "Asn"
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 11
<210> 12
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="His" or "Tyr" or "Phe"
<220>
<221> misc_feature
<222> (8)..(8)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (15)..(15)
<223> /replace="Tyr"
<220>
<221> misc_feature
<222> (15)..(15)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 12
<210> 13
<211> 11
<212> PRT
<213> Mus musculus
<400> 13
<210> 14
<211> 17
<212> PRT
<213> Mus musculus
<400> 14
<210> 15
<211> 10
<212> PRT
<213> Mus musculus
<400> 15
<210> 16
<211> 9
<212> PRT
<213> Mus musculus
<400> 16
<210> 17
<211> 7
<212> PRT
<213> Mus musculus
<400> 17
<210> 18
<211> 17
<212> PRT
<213> Mus musculus
<400> 18
<210> 19
<211> 120
<212> PRT
<213> Mus musculus
<400> 19
<210> 20
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 20
<210> 21
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 21
<210> 22
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 22
<210> 23
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 23
<210> 24
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 24
<210> 25
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 25
<210> 26
<211> 113
<212> PRT
<213> Mus musculus
<400> 26
<210> 27
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 27
<210> 28
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 28
<210> 29
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 29
<210> 30
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 30
<210> 31
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 31
<210> 32
<211> 11
<212> PRT
<213> Mus musculus
<400> 32
<210> 33
<211> 17
<212> PRT
<213> Mus musculus
<400> 33
<210> 34
<211> 10
<212> PRT
<213> Mus musculus
<400> 34
<210> 35
<211> 9
<212> PRT
<213> Mus musculus
<400> 35
<210> 36
<211> 7
<212> PRT
<213> Mus musculus
<400> 36
<210> 37
<211> 16
<212> PRT
<213> Mus musculus
<400> 37
<210> 38
<211> 120
<212> PRT
<213> Mus musculus
<400> 38
<210> 39
<211> 112
<212> PRT
<213> Mus musculus
<400> 39
<210> 40
<211> 7
<212> PRT
<213> Mus musculus
<400> 40
<210> 41
<211> 19
<212> PRT
<213> Mus musculus
<400> 41
<210> 42
<211> 10
<212> PRT
<213> Mus musculus
<400> 42
<210> 43
<211> 9
<212> PRT
<213> Mus musculus
<400> 43
<210> 44
<211> 17
<212> PRT
<213> Mus musculus
<400> 44
<210> 45
<211> 118
<212> PRT
<213> Mus musculus
<400> 45
<210> 46
<211> 113
<212> PRT
<213> Mus musculus
<400> 46
<210> 47
<211> 12
<212> PRT
<213> Mus musculus
<400> 47
<210> 48
<211> 16
<212> PRT
<213> Mus musculus
<400> 48
<210> 49
<211> 10
<212> PRT
<213> Mus musculus
<400> 49
<210> 50
<211> 9
<212> PRT
<213> Mus musculus
<400> 50
<210> 51
<211> 7
<212> PRT
<213> Mus musculus
<400> 51
<210> 52
<211> 17
<212> PRT
<213> Mus musculus
<400> 52
<210> 53
<211> 121
<212> PRT
<213> Mus musculus
<400> 53
<210> 54
<211> 113
<212> PRT
<213> Mus musculus
<400> 54
<210> 55
<211> 12
<212> PRT
<213> Mus musculus
<400> 55
<210> 56
<211> 17
<212> PRT
<213> Mus musculus
<400> 56
<210> 57
<211> 10
<212> PRT
<213> Mus musculus
<400> 57
<210> 58
<211> 9
<212> PRT
<213> Mus musculus
<400> 58
<210> 59
<211> 7
<212> PRT
<213> Mus musculus
<400> 59
<210> 60
<211> 16
<212> PRT
<213> Mus musculus
<400> 60
<210> 61
<211> 121
<212> PRT
<213> Mus musculus
<400> 61
<210> 62
<211> 112
<212> PRT
<213> Mus musculus
<400> 62
<210> 63
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (1)..(1)
<223> /replace="Phe"
<220>
<221> misc_feature
<222> (1)..(1)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace=" "
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="Ser"
<220>
<221> VARIANT
<222> (9)..(9)
<223> /replace="Pro"
<220>
<221> misc_feature
<222> (8)..(9)
<223> /note="Residues given in the sequence have no preference with respect to those
in the annotations for said positions"
<220>
<221> VARIANT
<222> (12)..(12)
<223> /replace="Tyr"
<220>
<221> misc_feature
<222> (12)..(12)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 63
<210> 64
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (2)..(2)
<223> /replace="Val"
<220>
<221> misc_feature
<222> (2)..(2)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (9)..(9)
<223> /replace="Ser"
<220>
<221> misc_feature
<222> (9)..(9)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (10)..(10)
<223> /replace="Asp"
<220>
<221> misc_feature
<222> (10)..(10)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 64
<210> 65
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (9)..(9)
<223> /replace="Met"
<220>
<221> misc_feature
<222> (9)..(9)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 65
<210> 66
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (2)..(2)
<223> /replace="Asn"
<220>
<221> VARIANT
<222> (3)..(3)
<223> /replace="Phe" or "Asp" or "His"
<220>
<221> misc_feature
<222> (2)..(3)
<223> /note="Residues given in the sequence have no preference with respect to those
in the annotations for said positions"
<220>
<221> VARIANT
<222> (4)..(4)
<223> /replace="Phe" or "His" or "Trp"
<220>
<221> misc_feature
<222> (4)..(4)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace="Phe" or "Thr" or "His"
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="Tyr" or "Phe" or "His" or "Leu"
<220>
<221> misc_feature
<222> (8)..(8)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 66
<210> 67
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (1)..(1)
<223> /replace="Gly"
<220>
<221> misc_feature
<222> (1)..(1)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (6)..(6)
<223> /replace="Asp" or "Gln" or "Asn"
<220>
<221> misc_feature
<222> (6)..(6)
<223> /note="Residue given in the sequence has no preference with respect to those
in the annotations for said position"
<400> 67
<210> 68
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (8)..(8)
<223> /replace="His" or "Tyr" or "Phe"
<220>
<221> VARIANT
<222> (9)..(9)
<223> /replace="Gly"
<220>
<221> misc_feature
<222> (8)..(9)
<223> /note="Residues given in the sequence have no preference with respect to those
in the annotations for said positions"
<220>
<221> VARIANT
<222> (11)..(11)
<223> /replace="Asp"
<220>
<221> misc_feature
<222> (11)..(11)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (13)..(13)
<223> /replace="Arg"
<220>
<221> misc_feature
<222> (13)..(13)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<220>
<221> VARIANT
<222> (15)..(15)
<223> /replace="Tyr"
<220>
<221> misc_feature
<222> (15)..(15)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 68
<210> 69
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (1)..(1)
<223> /replace="Met"
<220>
<221> misc_feature
<222> (1)..(1)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 69
<210> 70
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic consensus peptide"
<220>
<221> VARIANT
<222> (12)..(12)
<223> /replace="Glu"
<220>
<221> misc_feature
<222> (12)..(12)
<223> /note="Residue given in the sequence has no preference with respect to that
in the annotation for said position"
<400> 70
<210> 71
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 71
<210> 72
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 72
<210> 73
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 73
<210> 74
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 74
<210> 75
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 75
<210> 76
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 76
<210> 77
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 77
<210> 78
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 78
<210> 79
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 79
<210> 80
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 80
<210> 81
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 81
<210> 82
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 82
<210> 83
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 83
<210> 84
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 84
<210> 85
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 85
<210> 86
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 86
<210> 87
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 87
<210> 88
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 88
<210> 89
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 89
<210> 90
<211> 9
<212> PRT
<213> Mus musculus
<400> 90
<210> 91
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 91
<210> 92
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term biotin"
<400> 92
<210> 93
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term KLH"
<400> 93
<210> 94
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="C-term KLH"
<400> 94
<210> 95
<211> 5
<212> PRT
<213> Unknown
<220>
<221> source
<223> /note="Description of Unknown: Bradykinin 1-5 peptide"
<400> 95
<210> 96
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 96
<210> 97
<211> 136
<212> PRT
<213> Mus musculus
<400> 97
<210> 98
<211> 134
<212> PRT
<213> Mus musculus
<400> 98
<210> 99
<211> 136
<212> PRT
<213> Mus musculus
<400> 99
<210> 100
<211> 137
<212> PRT
<213> Mus musculus
<400> 100
<210> 101
<211> 137
<212> PRT
<213> Mus musculus
<400> 101
<210> 102
<211> 133
<212> PRT
<213> Mus musculus
<400> 102
<210> 103
<211> 134
<212> PRT
<213> Mus musculus
<400> 103
<210> 104
<211> 134
<212> PRT
<213> Mus musculus
<400> 104
<210> 105
<211> 133
<212> PRT
<213> Mus musculus
<400> 105
<210> 106
<211> 133
<212> PRT
<213> Mus musculus
<400> 106
<210> 107
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 107
aaaagcaggc ttaggagcgg ccgccatggc gtcccaggcc tcgctg 46
<210> 108
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic primer"
<400> 108
caagaaagct gggtcggatc cttataaagt tcccagaacc ctggtc 46
<210> 109
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic oligonucleotide"
<400> 109
gcatacccat acgacgtccc agactacgct 30
<210> 110
<211> 1005
<212> DNA
<213> Mus musculus
<400> 110
<210> 111
<211> 111
<212> PRT
<213> Mus musculus
<400> 111
<210> 112
<211> 105
<212> PRT
<213> Homo sapiens
<400> 112
<210> 113
<211> 120
<212> PRT
<213> Mus musculus
<400> 113
<210> 114
<211> 123
<212> PRT
<213> Homo sapiens
<400> 114
<210> 115
<211> 113
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 115
<210> 116
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 116
<210> 117
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<220>
<221> source
<223> /note="N-term biotin"
<400> 117
<210> 118
<211> 127
<212> PRT
<213> Mus musculus
<400> 118
<210> 119
<211> 125
<212> PRT
<213> Mus musculus
<400> 119
<210> 120
<211> 127
<212> PRT
<213> Mus musculus
<400> 120
<210> 121
<211> 128
<212> PRT
<213> Mus musculus
<400> 121
<210> 122
<211> 128
<212> PRT
<213> Mus musculus
<400> 122
<210> 123
<211> 119
<212> PRT
<213> Mus musculus
<400> 123
<210> 124
<211> 120
<212> PRT
<213> Mus musculus
<400> 124
<210> 125
<211> 120
<212> PRT
<213> Mus musculus
<400> 125
<210> 126
<211> 120
<212> PRT
<213> Mus musculus
<400> 126
<210> 127
<211> 360
<212> DNA
<213> Mus musculus
<400> 127
<210> 128
<211> 339
<212> DNA
<213> Mus musculus
<400> 128
<210> 129
<211> 360
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polynucleotide"
<400> 129
<210> 130
<211> 339
<212> DNA
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polynucleotide"
<400> 130
<210> 131
<211> 119
<212> PRT
<213> Mus musculus
<400> 131
<210> 132
<211> 4
<212> PRT
<213> Homo sapiens
<400> 132
<210> 133
<211> 6
<212> PRT
<213> Homo sapiens
<400> 133
<210> 134
<211> 220
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 134
<210> 135
<211> 229
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic polypeptide"
<400> 135
<210> 136
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic peptide"
<400> 136
<210> 137
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<221> source
<223> /note="Description of Artificial Sequence: Synthetic 6xHis tag"
<400> 137
i) a heavy chain variable domain comprising a heavy chain complementarity determining region 3 (HCDR3) amino acid sequence selected from the group consisting of:
a) SEQ ID NO: 7 [X1Y X2 X3D X4HAM X5Y], wherein
X1 is Y, F or H,
X2 is R, D, A, V, L, I, M, F, Y or W,
X3 is Y, F, W or H,
X4 is D, E or Y, and
X5 is D or E;
b) SEQ ID NO: 63 [X1EYDGX2YX3X4LDX5], wherein
X1 is W or F,
X2 is N or no amino acid,
X3 is Y or S,
X4 is D or P, and
X5 is F or Y;
c) SEQ ID NO: 13;
d) SEQ ID NO: 32;
e) SEQ ID NO: 40;
f) SEQ ID NO: 47; and
g) SEQ ID NO: 55;
ii) a heavy chain variable domain comprising a heavy chain complementarity determining region 2 (HCDR2) amino acid sequence selected from the group consisting of:
h) SEQ ID NO: 8 [YFX1PX2NGNTGYNQKFRG], wherein
X1 is D, R, A, V, L, I, M, F, Y or W, and
X2 is Y, D, E, N, or Q;
i) SEQ ID NO: 64 [WX1DPENGDX2X3YAPKFQG], wherein
X1 is I, or V,
X2 is T, or S, and
X3 is G, or D;
j) SEQ ID NO: 14;
k) SEQ ID NO: 33;
l) SEQ ID NO: 41;
m) SEQ ID NO: 48; and
n) SEQ ID NO: 56;
iii) a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (HCDR1) amino acid sequence selected from the group consisting of:
o) SEQ ID NO: 9 [GYSFTDYX1IY], wherein X1 is N, W or Y;
p) SEQ ID NO: 65 [GFNIKDYYX1H], wherein X1 is L, or M;
q) SEQ ID NO: 15;
r) SEQ ID NO: 34;
s) SEQ ID NO: 42;
t) SEQ ID NO: 49; and
u) SEQ ID NO: 57;
iv) a light chain variable domain comprising a light chain complementarity determining region 3 (LCDR3) amino acid sequence selected from the group consisting of:
v) SEQ ID NO: 10 [QQ X1 X2S X3P X4T], wherein
X1 is Y, F or H,
X2 is Y, F, H or W,
X3 is Y, F, T or H, and
X4 is W, Y, F, H or L;
w) SEQ ID NO: 66 [QX1X2X3SX4PX5T], wherein
X1 is Q or N,
X2 is Y, F, D or H,
X3 is Y, F, H or W,
X4 is Y, F, T or H, and
X5 is W, Y, F, H or L;
x) SEQ ID NO: 69 [X1QGTHFPYT], wherein X1 is L or M;
y) SEQ ID NO: 16;
z) SEQ ID NO: 35;
aa) SEQ ID NO: 43;
bb) SEQ ID NO: 50; and
cc) SEQ ID NO: 58, or
v) a light chain variable domain comprising a light chain complementarity determining region 2 (LCDR2) amino acid sequence selected from the group consisting of:
dd) SEQ ID NO: 11 [WASTRX1], wherein X1 is E, D, Q or N;
ee) SEQ ID NO: 67 [X1ASTRX2], wherein
X1 is W or G, and
X2 is E, D, Q or N;
ff) SEQ ID NO: 17;
gg) SEQ ID NO: 36;
hh) SEQ ID NO: 51; and
ii) SEQ ID NO: 59;
andvi) a light chain variable domain comprising a light chain complementarity determining region 1 (LCDR1) amino acid sequence selected from the group consisting of:
jj) SEQ ID NO: 12 [KSSQSLL X1SSNQKN X2LA], wherein
X1 is W, H, Y or F, and
X2 is H or Y;
kk) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], wherein
X1 is W, H, Y or F,
X2 is S or G,
X3 is N or D,
X4 is K or R,
X5 is H or Y;
ll) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN], wherein X1 is K or E;
mm) SEQ ID NO: 18;
nn) SEQ ID NO: 37;
oo) SEQ ID NO: 44;
pp) SEQ ID NO: 52; and
qq) SEQ ID NO: 60.
a) a heavy chain variable domain comprising the consensus HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively; and
b) a light chain variable domain comprising the consensus LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 32, 33, and 34, respectively; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 35, 36, and 37, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 40, 41 and 42, respectively; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 43, 17, and 44, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 47, 48, and 49, respectively; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 50, 51, and 52, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively.
a) a heavy chain variable domain comprising the HCDR3, HCDR2 and HCDR1 region amino sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and one or more amino acid substitution at positions selected from the group consisting of H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, and H108, according to Kabat; and
b) a light chain variable domain comprising the LCDR3, LCDR2 and LCDR1 region amino sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and one or more amino acid substitution at positions selected from the group consisting of L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 and L104, according to Kabat.
administering to a subject in need thereof a pharmaceutical composition comprising the antibody or antigen binding fragment thereof and one or more pharmaceutically acceptable carriers,
wherein the antibody or antigen binding fragment thereof comprises the heavy chain and light chain variable domain amino acid sequences set forth in SEQ ID NOs: 24 and 30, respectively.
administering to a subject in need thereof a pharmaceutical composition comprising the antibody or antigen binding fragment thereof and one or more pharmaceutically acceptable carriers,
wherein the antibody or antigen binding fragment thereof comprises:
a) a heavy chain variable region domain amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61; and
b) a light chain variable domain amino acid sequence with at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 39, 46, 54, and 62.
administering to a subject in need thereof a pharmaceutical composition comprising the antibody or antigen binding fragment thereof and one or more pharmaceutically acceptable carriers,
wherein the antibody or antigen binding fragment thereof comprises:
a) a heavy chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 24, 25, 38, 45, 53, and 61; and
b) a light chain variable domain amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30, 31, 39, 46, 54, and 62.
administering to a subject in need thereof a pharmaceutical composition comprising the antibody or antigen binding fragment thereof and one or more pharmaceutically acceptable carriers,
wherein the antibody or antigen binding fragment thereof competes for binding to Kallidin and des-Arg10-Kallidin with an antibody comprising the heavy chain and light chain variable region amino acid sequences set forth in SEQ ID NOs: 19 and 26, SEQ ID NOs: 38 and 39, SEQ ID NOs: 45 and 46, SEQ ID NOs: 53 and 54, or SEQ ID NOs: 61 and 62, respectively.
i) eine variable Domäne der schweren Kette, umfassend eine HCDR3-Aminosäuresequenz (Heavy Chain Complementarity Determining Region 3), die aus der nachfolgenden Gruppe gewählt ist:
a) SEQ ID NR.: 7 [X1Y X2 X3D X4HAM X5Y], wobei
X1 Y, F oder H ist,
X2 R, D, A, V, L, I, M, F, Y oder W ist,
X3 Y, F, W oder H ist,
X4 D, E oder Y ist und
X5 D oder E ist,
b) SEQ ID NR.: 63 [X1EYDGX2YX3X4LDX5], wobei
X1 W oder F ist,
X2 N oder keine Aminosäure ist,
X3 Y oder S ist,
X4 D oder P ist und
X5 F oder Y ist,
c) SEQ ID NR.: 13;
d) SEQ ID NR.: 32;
e) SEQ ID NR.: 40;
f) SEQ ID NR.: 47 und
g) SEQ ID NR.: 55;
ii) eine variable Domäne der schweren Kette, umfassend eine HCDR2-Aminosäuresequenz, die aus der nachfolgenden Gruppe gewählt ist:
h) SEQ ID NR.: 8 [YFX1PX2NGNTGYNQKFRG], wobei
X1 D, R, A, V, L, I, M, F, Y oder W ist und
X2 Y, D, E, N oder Q ist;
i) SEQ ID NR.: 64 [WX1DPENGDX2X3YAPKFQG], wobei
X1 I oder V ist,
X2 T oder S ist und
X3 G oder D ist
j) SEQ ID NR.: 14;
k) SEQ ID NR.: 33;
l) SEQ ID NR.: 41;
m) SEQ ID NR.: 48 und
n) SEQ ID NR.: 56;
iii) eine variable Domäne der schweren Kette, umfassend eine HCDR1-Aminosäuresequenz, die aus der nachfolgenden Gruppe gewählt ist:
o) SEQ ID NR.: 9 [GYSFTDYX1IY], wobei X1 N, W oder Y ist;
p) SEQ ID NR.: 65 [GFNIKDYYX1H], wobei X1 L oder M ist,
q) SEQ ID NR.: 15;
r) SEQ ID NR.: 34;
s) SEQ ID NR.: 42;
t) SEQ ID NR.: 49 und
u) SEQ ID NR.: 57;
iv) eine variable Domäne der leichten Kette, umfassend eine LCDR3-Aminosäuresequenz (Light Chain Complementarity Determining Region 3), die aus der nachfolgenden Gruppe gewählt ist:
v) SEQ ID NR.: 10 [QQ X1 X2S X3P X4T], wobei
X1 Y, F oder H ist,
X2 Y, F, H oder W ist,
X3 Y, F, T oder H ist und X4 W, Y, F, H oder L ist,
w) SEQ ID NR.: 66 [QX1X2X3SX4PX5T], wobei
X1 Q oder N ist,
X2 Y, F, D oder H ist,
X3 Y, F, H oder W ist,
X4 Y, F, T oder H ist und
X5 W, Y, F, H oder L ist,
x) SEQ ID NR.: 69 [X1QGTHFPYT], wobei X1 L oder M ist,
y) SEQ ID NR.: 16;
z) SEQ ID NR.: 35;
aa) SEQ ID NR.: 43;
bb) SEQ ID NR.: 50 und
cc) SEQ ID NR.: 58 oder
v) eine variable Domäne der leichten Kette, umfassend eine LCDR2-Aminosäuresequenz, die aus der nachfolgenden Gruppe gewählt ist:
dd) SEQ ID NR: 11 [WASTRX1], wobei X1 E, D, Q oder N ist;
ee) SEQ ID NR: 67 [X1ASTRX2], wobei
X1 W oder G ist und
X2 E, D, Q oder N ist,
ff) SEQ ID NR: 17;
gg) SEQ ID NR: 36;
hh) SEQ ID NR: 51 und
ii) SEQ ID NR: 59;
undvi) eine variable Domäne der leichten Kette, umfassend eine LCDR1-Aminosäuresequenz, die aus der nachfolgenden Gruppe gewählt ist:
jj) SEQ ID NR: 12 [KSSQSLL X1SSNQKN X2LA], wobei
X1 W, H, Y oder F ist und
X2 H oder Y ist;
kk) SEQ ID NR: 68 [KSSQSLLX1X2SX3QX4NX5LA], wobei
X1 W, H, Y oder F ist,
X2 S oder G ist,
X3 N oder D ist
X4 K oder R ist,
X5 H oder Y ist,
ll) SEQ ID NR: 70 [KSSQSLLYSNGX1TYLN], wobei X1 K oder E ist,
mm) SEQ ID NR: 18;
nn) SEQ ID NR: 37;
oo) SEQ ID NR: 44;
pp) SEQ ID NR: 52 und
qq) SEQ ID NR: 60.
a) eine variable Domäne der schweren Kette, umfassend die Konsens-Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 7, 8 bzw. 9 und
b) eine variable Domäne der leichten Kette, umfassend die Konsens-Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 10, 11 bzw. 12.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 13, 14 bzw. 15 und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 16, 17 bzw. 18.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 32, 33 bzw. 34 und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 35, 36 bzw. 37.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 40, 41 bzw. 42 und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 43, 17 bzw. 44.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 47, 48 bzw. 49 und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 50, 51 bzw. 52.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 55, 56 bzw. 57 und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 58, 59 bzw. 60.
a) eine variable Domäne der schweren Kette, umfassend Aminosäuresequenzen der Regionen HCDR3, HCDR2 und HCDR1 nach SEQ ID NR: 13, 14 bzw. 15 sowie mindestens eine Aminosäuresubstitution in den aus der nachfolgenden Gruppe gewählten Positionen: H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87 und H108 nach Rabat, und
b) eine variable Domäne der leichten Kette, umfassend die Aminosäuresequenzen der Regionen LCDR3, LCDR2 und LCDR1 nach SEQ ID NR: 16, 17 bzw. 18 und mindestens eine Aminosäuresubstitution in den aus der nachfolgenden Gruppe gewählten Positionen: L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 und L104 nach Rabat.
Verabreichen einer den Antikörper bzw. das antigenbindende Fragmentdavon sowie mindestens einen pharmazeutisch unbedenklichen Träger umfassenden pharmazeutischen Zusammensetzung an einen behandlungsbedürftigen Probanden,
wobei der Antikörper bzw. das antigenbindende Fragment davon die Aminosäuresequenzen der variablen Domänen der schweren und leichten Kette nach SEQ ID NR: 24 bzw. 30 umfasst.
Verabreichen einer den Antikörper bzw. das antigenbindende Fragmentdavon sowie mindestens einen pharmazeutisch unbedenklichen Träger umfassenden pharmazeutischen Zusammensetzung an einen behandlungsbedürftigen Probanden,
wobei der Antikörper bzw. das antigenbindende Fragment davon umfasst:
a) eine Aminosäuresequenz der variablen Region der schweren Kette mit mindestens 90 % Identität mit einer aus der nachfolgenden Gruppe gewählten Aminosäuresequenz: SEQ ID NR: 19, 20, 21, 22, 24, 25, 38, 45, 53 und 61 und
b) eine Aminosäuresequenz der variablen Domäne der leichten Kette mit mindestens 90 % Identität mit einer aus der nachfolgenden Gruppe gewählten Aminosäuresequenz: SEQ ID NR: 26, 27, 28, 29, 30, 31, 39, 46, 54 und 62.
Verabreichen einer den Antikörper bzw. das antigenbindende Fragmentdavon sowie mindestens einen pharmazeutisch unbedenklichen Träger umfassenden pharmazeutischen Zusammensetzung an einen behandlungsbedürftigen Probanden,
wobei der Antikörper bzw. das antigenbindende Fragment davon umfasst:
a) eine variable Domäne der schweren Kette, umfassend eine aus der nachfolgenden Gruppe gewählte Aminosäuresequenz: SEQ ID NR: 19, 20, 21, 22, 24, 25, 38, 45, 53 und 61 und
b) eine aus der nachfolgenden Gruppe gewählte Aminosäuresequenz der variablen Domäne der leichten Kette: SEQ ID NR: 26, 27, 28, 29, 30, 31, 39, 46, 54 und 62.
Verabreichen einer den Antikörper bzw. das antigenbindende Fragmentdavon sowie mindestens einen pharmazeutisch unbedenklichen Träger umfassenden pharmazeutischen Zusammensetzung an einen behandlungsbedürftigen Probanden,
wobei der Antikörper bzw. das antigenbindende Fragment davon um die Bindung an Kallidin und Des-Arg10-Kallidin mit einem Antikörper konkurriert, der jeweils die Aminosäuresequenzen der variablen Regionen der schweren und leichten Kette nach SEQ ID NR: 19 und 26, SEQ ID NR: 38 und 39, SEQ ID NR: 45 und 46, SEQ ID NR: 53 und 54, oder SEQ ID NR: 61 und 62 umfasst.
i) un domaine variable de chaîne lourde comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne lourde 3 (HCDR3) sélectionnée dans le groupe consistant en :
a) SEQ ID NO: 7 [X1YX2X3DX4HAMX5Y], dans laquelle
X1 est Y, F ou H,
X2 est R, D, A, V, L, I, M, F, Y ou W,
X3 est Y, F, W ou H,
X4 est D, E ou Y, et,
X5 est D ou E ;
b) SEQ ID NO: 63 [X1EYDGX2YX3X4LDX5], dans laquelle
X1 est W ou F,
X2 est N ou aucun acide aminé,
X3 est Y ou S,
X4 est D ou P, et
X5 est F ou Y ;
c) SEQ ID NO: 13;
d) SEQ ID NO: 32 ;
e) SEQ ID NO: 40 ;
f) SEQ ID NO: 47 ; et
g) SEQ ID NO: 55 ;
ii) un domaine variable de chaîne lourde comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne lourde 2 (HCDR2) sélectionnée dans le groupe consistant en :
h) SEQ ID NO: 8 [YFX1PX2NGNTGYNQKFRG], dans laquelle
X1 est D, R, A, V, L, I, M, F, Y ou W, et
X2 est Y, D, E, N, ou Q ;
i) SEQ ID NO: 64 [WX1DPENGDX2X3YAPKFQG], dans laquelle
X1 est I, ou V,
X2 est T, ou S, et
X3 est G, ou D ;
j) SEQ ID NO: 14 ;
k) SEQ ID NO: 33 ;
l) SEQ ID NO: 41 ;
m) SEQ ID NO: 48 ; et
n) SEQ ID NO: 56 ;
iii) un domaine variable de chaîne lourde comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne lourde 1 (HCDR1) sélectionnée dans le groupe consistant en :
o) SEQ ID NO: 9 [GYSFTDYX1IY], dans laquelle X1 est N, W ou Y ;
p) SEQ ID NO: 65 [GFNIKDYYX1H], dans laquelle X1 est L, ou M ;
q) SEQ ID NO: 15 ;
r) SEQ ID NO: 34 ;
s) SEQ ID NO: 42 ;
t) SEQ ID NO: 49 ; et
u) SEQ ID NO: 57 ;
iv) un domaine variable de chaîne légère comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne légère 3 (LCDR3) sélectionnée dans le groupe consistant en :
v) SEQ ID NO: 10 [QQX1X2SX3PX4T], dans laquelle
X1 est Y, F ou H,
X2 est Y, F, H ou W,
X3 est Y, F, T ou H, et,
X4 est W, Y, F, H ou L ;
w) SEQ ID NO: 66 [QX1X2X3SX4PX5T], dans laquelle
X1 est Q ou N,
X2 est Y, F, D ou H,
X3 est Y, F, H ou W,
X4 est Y, F, T ou H, et
X5 est W, Y, F, H ou L ;
x) SEQ ID NO: 69 [X1QGTHFPYT], dans laquelle X1 est L ou M ;
y) SEQ ID NO: 16;
z) SEQ ID NO: 35 ;
aa) SEQ ID NO: 43 ;
bb) SEQ ID NO: 50 ; et
cc) SEQ ID NO: 58, ou
v) un domaine variable de chaîne légère comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne légère 2 (LCDR2) sélectionnée dans le groupe consistant en :
dd) SEQ ID NO: 11 [WASTRX1], dans laquelle X1 est E, D, Q ou N ;
ee) SEQ ID NO: 67 [X2ASTRX2], dans laquelle
X1 est W ou G, et
X2 est E, D, Q ou N ;
ff) SEQ ID NO: 17 ;
gg) SEQ ID NO: 36 ;
hh) SEQ ID NO: 51 ; et
ii) SEQ ID NO: 59 ;
etvi) un domaine variable de chaîne légère comprenant une séquence d'acides aminés d'une région déterminant la complémentarité de chaîne légère 1 (LCDR1) sélectionnée dans le groupe consistant en :
jj) SEQ ID NO: 12 [KSSQSLLX1SSNQKNX2LA], dans laquelle
X1 est W, H, Y ou F, et
X2 est H ou Y ;
kk) SEQ ID NO: 68 [KSSQSLLX1X2SX3QX4NX5LA], dans laquelle
X1 est W, H, Y ou F,
X2 est S ou G,
X3 est N ou D,
X4 est K ou R,
X5 est H ou Y,
ll) SEQ ID NO: 70 [KSSQSLLYSNGX1TYLN], dans laquelle X1 est K ou E ;
mm) SEQ ID NO: 18 ;
nn) SEQ ID NO: 37 ;
oo) SEQ ID NO: 44 ;
pp) SEQ ID NO: 52 ; et
qq) SEQ ID NO: 60.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 consensus décrites dans SEQ ID NO: 7, 8, et 9, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 consensus décrites dans SEQ ID NO: 10, 11, et 12, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 13, 14, et 15, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 16, 17, et 18, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 32, 33, et 34, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 35, 36, et 37, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 40, 41, et 42, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 43, 17, et 44, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 47, 48, et 49, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 50, 51, et 52, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 55, 56, et 57, respectivement ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 58, 59, et 60, respectivement.
a) un domaine variable de chaîne lourde comprenant les séquences d'acides aminés des régions HCDR3, HCDR2 et HCDR1 décrites dans SEQ ID NO: 13, 14, et 15, respectivement, et une ou plusieurs substitutions d'acides aminés à des positions sélectionnées dans le groupe consistant en H1, H5, H9, H11, H12, H16, H38, H40, H41, H43, H44, H66, H75, H79, H81, H82A, H83, H87, et H108, selon Kabat ; et
b) un domaine variable de chaîne légère comprenant les séquences d'acides aminés des régions LCDR3, LCDR2 et LCDR1 décrites dans SEQ ID NO: 16, 17, et 18, respectivement, et une ou plusieurs substitutions d'acides aminés à des positions sélectionnées dans le groupe consistant en L5, L9, L15, L18, L19, L21, L22, L43, L63, L78, L79, L83, L85, L100 et L104, selon Kabat.
l'administration, à un sujet en ayant besoin, d'une composition pharmaceutique comprenant l'anticorps ou fragment de celui-ci se liant à un antigène et un ou plusieurs véhicules pharmaceutiquement acceptables,
où l'anticorps ou fragment de celui-ci se liant à un antigène comprend les séquences d'acides aminés des domaines variables de chaîne lourde et chaîne légère décrites dans SEQ ID NO: 24 et 30, respectivement.
l'administration, à un sujet en ayant besoin, d'une composition pharmaceutique comprenant l'anticorps ou fragment de celui-ci se liant à un antigène et un ou plusieurs véhicules pharmaceutiquement acceptables,
où l'anticorps ou fragment de celui-ci se liant à un antigène comprend :
a) une séquence d'acides aminés de domaine de région variable de chaîne lourde présentant au moins 90 % d'identité avec une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NO: 19, 20, 21, 22, 24, 25, 38, 45, 53, et 61 ; et
b) une séquence d'acides aminés de domaine variable de chaîne légère présentant au moins 90 % d'identité avec une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NO: 26, 27, 28, 29, 30, 31, 39, 46, 54, et 62.
l'administration, à un sujet en ayant besoin, d'une composition pharmaceutique comprenant l'anticorps ou fragment de celui-ci se liant à un antigène et un ou plusieurs véhicules pharmaceutiquement acceptables,
où l'anticorps ou fragment de celui-ci se liant à un antigène comprend :
a) un domaine variable de chaîne lourde comprenant une séquence d'acides aminés sélectionnée dans le groupe consistant en SEQ ID NO: 19, 20, 21, 22, 24, 25, 38, 45, 53, et 61 ; et
b) une séquence d'acides aminés de domaine variable de chaîne légère sélectionnée dans le groupe consistant en SEQ ID NO: 26, 27, 28, 29, 30, 31, 39, 46, 54, et 62.
l'administration, à un sujet en ayant besoin, d'une composition pharmaceutique comprenant l'anticorps ou fragment de celui-ci se liant à un antigène et un ou plusieurs véhicules pharmaceutiquement acceptables,
où l'anticorps ou fragment de celui-ci se liant à un antigène entre en compétition pour une liaison à la kallidine et la des-Arg10-kallidine avec un anticorps comprenant les séquences d'acides aminés des régions variables de chaîne lourde et chaîne légère décrites dans SEQ ID NO: 19 et 26, SEQ ID NO: 38 et 39, SEQ ID NO: 45 et 46, SEQ ID NO: 53 et 54, ou SEQ ID NO: 61 et 62, respectivement.
REFERENCES CITED IN THE DESCRIPTION
Patent documents cited in the description
Non-patent literature cited in the description